RESUMO
BACKGROUND & AIMS: Alagille syndrome is a genetic disorder characterized by cholestasis, ocular abnormalities, characteristic facial features, heart defects, and vertebral malformations. Most cases are associated with mutations in JAGGED1 (JAG1), which encodes a Notch ligand, although it is not clear how these contribute to disease development. We aimed to develop a mouse model of Alagille syndrome to elucidate these mechanisms. METHODS: Mice with a missense mutation (H268Q) in Jag1 (Jag1+/Ndr mice) were outbred to a C3H/C57bl6 background to generate a mouse model for Alagille syndrome (Jag1Ndr/Ndr mice). Liver tissues were collected at different timepoints during development, analyzed by histology, and liver organoids were cultured and analyzed. We performed transcriptome analysis of Jag1Ndr/Ndr livers and livers from patients with Alagille syndrome, cross-referenced to the Human Protein Atlas, to identify commonly dysregulated pathways and biliary markers. We used species-specific transcriptome separation and ligand-receptor interaction assays to measure Notch signaling and the ability of JAG1Ndr to bind or activate Notch receptors. We studied signaling of JAG1 and JAG1Ndr via NOTCH 1, NOTCH2, and NOTCH3 and resulting gene expression patterns in parental and NOTCH1-expressing C2C12 cell lines. RESULTS: Jag1Ndr/Ndr mice had many features of Alagille syndrome, including eye, heart, and liver defects. Bile duct differentiation, morphogenesis, and function were dysregulated in newborn Jag1Ndr/Ndr mice, with aberrations in cholangiocyte polarity, but these defects improved in adult mice. Jag1Ndr/Ndr liver organoids collapsed in culture, indicating structural instability. Whole-transcriptome sequence analyses of liver tissues from mice and patients with Alagille syndrome identified dysregulated genes encoding proteins enriched at the apical side of cholangiocytes, including CFTR and SLC5A1, as well as reduced expression of IGF1. Exposure of Notch-expressing cells to JAG1Ndr, compared with JAG1, led to hypomorphic Notch signaling, based on transcriptome analysis. JAG1-expressing cells, but not JAG1Ndr-expressing cells, bound soluble Notch1 extracellular domain, quantified by flow cytometry. However, JAG1 and JAG1Ndr cells each bound NOTCH2, and signaling from NOTCH2 signaling was reduced but not completely inhibited, in response to JAG1Ndr compared with JAG1. CONCLUSIONS: In mice, expression of a missense mutant of Jag1 (Jag1Ndr) disrupts bile duct development and recapitulates Alagille syndrome phenotypes in heart, eye, and craniofacial dysmorphology. JAG1Ndr does not bind NOTCH1, but binds NOTCH2, and elicits hypomorphic signaling. This mouse model can be used to study other features of Alagille syndrome and organ development.
Assuntos
Síndrome de Alagille/genética , Proteína Jagged-1/genética , Mutação de Sentido Incorreto , Síndrome de Alagille/metabolismo , Síndrome de Alagille/patologia , Animais , Ductos Biliares Intra-Hepáticos/metabolismo , Ductos Biliares Intra-Hepáticos/patologia , Diferenciação Celular , Técnicas de Cocultura , Modelos Animais de Doenças , Feminino , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica no Desenvolvimento , Predisposição Genética para Doença , Células HEK293 , Humanos , Proteína Jagged-1/metabolismo , Masculino , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Morfogênese , Organoides , Fenótipo , Receptor Notch2/genética , Receptor Notch2/metabolismo , Transdução de Sinais , TransfecçãoRESUMO
The signaling adaptor protein Crk has been shown to play an important role in various human cancers. However, its regulatory machinery is not clear. Here, we demonstrated that Crk induced EMT in A549 human lung adenocarcinoma cells through differential regulation of Rac1/Snail and RhoA/Slug, leading to decreased expression of E-cadherin and increased N-cadherin, fibronectin, and MMP2 expression. Cancer cells with mesenchymal features produced TGF-ß and also increased the levels of TGF-ß receptor. TGF-ß increased the endogenous levels of Crk and also augmented Crk-dependent expression of Snail and Slug, and conversely TGF-ß receptor inhibitor suppressed the levels of Snail and Slug. Overexpression of Crk was observed at the invasive front of human lung cancer tissues and was significantly associated with poor prognosis. Thus, TGF-ß and Crk collaborate to form a positive feedback loop to facilitate EMT, which may lead to the malignancy of human cancers possibly being affected by their microenvironment.
Assuntos
Transição Epitelial-Mesenquimal/genética , Neoplasias Pulmonares/genética , Proteínas Proto-Oncogênicas c-crk/genética , Transdução de Sinais/genética , Fator de Crescimento Transformador beta/genética , Células A549 , Animais , Sequência de Bases , Linhagem Celular Tumoral , Células Cultivadas , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos Nus , Proteínas Proto-Oncogênicas c-crk/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Transplante HeterólogoRESUMO
BACKGROUND: Ionizing irradiation is an effective treatment for malignant glioma (MG); however, a higher rate of recurrence with more aggressive phenotypes is a vital issue. Although epithelial-mesenchymal transition (EMT) is involved in irradiation-induced cancer progression, the role for such phenotypic transition in MG remains unknown. METHODS: To investigate the mechanism of irradiation-dependent tumor progression in MG, we performed immunohistochemistry (IHC) and qRT-PCR using primary and recurrent MG specimens, MG cell lines, and primary culture cells of MG. siRNA technique was used for MG cell lines. RESULTS: In 22 cases of clinically recurrent MG, the expression of the mesenchymal markers vimentin and CD44 was found to be increased by IHC. In paired identical MG of 7 patients, the expression of collagen, MMPs, and YKL-40 were also elevated in the recurrent MGs, suggesting the The Cancer Genome Atlas-based mesenchymal subtype. Among EMT regulators, sustained elevation of Snail was observed in MG cells at 21 days after irradiation. Cells exhibited an upregulation of migration, invasion, numbers of focal adhesion, and MMP-2 production, and all of these mesenchymal features were abrogated by Snail knockdown. Intriguingly, phosphorylation of ERK1/2 and GSK-3ß were increased after irradiation in a Snail-dependent manner, and TGF-ß was elevated in both fibroblasts and macrophages but not in MG cells after irradiation. It was noteworthy that irradiated cells also expressed stemness features such as SOX2 expression and tumor-forming potential in vivo. CONCLUSIONS: We here propose a novel concept of glial-mesenchymal transition after irradiation in which the sustained Snail expression plays an essential role.